Stable near-infrared (NIR) marker dyes based on benzopyrylium-polymethines

ABSTRACT

The disclosure is directed to so-called laser-compatible NIR marker dyes based on polymethines for use in optical, in particular, fluorescence optical determination and detection methods, for example, in the fields of medicine, pharmaceutics and in the areas of life science, materials science and environmental science. The disclosure further discusses the aim of the invention which was to create NIR marker dyes based on polymethine which have a high degree of photostability and stability in storage as well as a high fluorescent yield and which can be excited to fluorescence in the easiest possible manner by means of laser radiation in the visible or NIR spectral range, particularly with light of an argon, helium/neon, or diode laser. Dyes based on polymethine of general formula (I) are used.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority of International Application No.PCT/DE01/01946, filed May 22, 2001 and German Application No. 100 25820.4, filed May 23, 2000 and No. 200 22 277.5, filed May 23, 2000, thecomplete disclosures of which are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

a) Field of the Invention

The invention is directed to so-called laser-compatible NIR marker dyesbased on polymethines for use in optical, in particular, fluorescenceoptical determination and detection methods. Typical applications of theprocess are based on the reaction of dye marked antigens, antibodies,ligands or DNA segments with the respective complementary species.

Possible uses exist, for example, in the fields of medicine,pharmaceutics and in the areas of life science, materials science, inenvironmental monitoring and in the detection of organic and inorganicmicro-samples occurring naturally and in technological contexts, butthey are not limited to the aforementioned fields.

b) Description of the Related Art

The usability of polymethines as NIR markers has been known of for along time; they distinguish themselves by their strong absorption maximawhich can easily be transposed into the NIR range (Fabian, J.; Nakazumi,H.; Matsuoka, M.: Chem. Rev. 1992, 92, 1197). With a suitablesubstituent pattern and pi-electron system and at a sufficient quantumyield they also fluoresce in the red and near infrared (NIR) range.Correspondingly, these compounds are widely used in differenttechnological fields: as sensitizers in AgX materials, as laser dyes, asquantum counters, as indicator dyes in sensor technology, as lightabsorbers in writable CDs and last but not least as biomarkers(“Near-Infrared Dyes for High Technology Application”, published byDaehne, S.; Resch-Genger, U.; Wolfbeis, O.-S., Kluwer, AcademicPublishers—Dordrecht/Boston/London—1998).

The number of polymethines used as biomarkers is limited. So far, onlythe trimethine Cy3 derived from astraphloxine (DE 410 487), thevinylogous pentamethine Cy5 and the doubly vinylogous heptamethine Cy7with absorption maxima at approximately 550 nm, approximately 650 nm andapproximately 750 nm have so far found wide commercial application inthis manner (U.S. Pat. No. 5,627,027). Also available are thepolysulfonated trimethine Cy3.5 derived from the commercial hepatmethine“Indocynaninegreen” or “Cardio Green” and the pentamethine Cy5.5 (U.S.Pat. No. 5,569,766). Heptamethines with aliphatic bridges in thepolymethine chain have been developed by Patonay (U.S. Pat. No.5,800,995). All commercial biomarkers are characterized by terminalheteroaromatics derived from indene or heteroindene (Fischer's base). Ifmethylsubstituted cycloimmonium salts are used as terminal polymethinebuilding blocks, it is necessary to arrange at least five sequential sp²hybridized carbon atoms (pentamethines) between the heterocycles togenerate absorption maxima at the boundary to the NIR range.

The NIR polymethines used in technology as biomarkers have the distinctdisadvantage that lengthening the polymethine chain increases theopportunities for nucleophilic or electrophilic attack on the chain, inconsequence of which the pi-system is destroyed. Further disadvantagesof these marker dyes consist in their insufficient photostability andstability in storage, complicated synthesis and purification stages, lowabsorption coefficients/low fluorescent quantum yields as well asundesired changes of their optical properties in the presence of orafter bonding with proteins or nucleic acid oligomeres. For example, areduction of the fluorescent quantum yield of Cy5 has been described forthe covalent bonding with different albumins (Oswald, B.; Patsenker, L.;Duschl, J.; Szmacinski, H.; Wolfbeis, O. S.; Terpeschnig, E.;Bioconjugate Chem. 1999, 10, 925-931).

The use of pyrylium and benzopyrylium heterocycles or the correspondingmesomeric chromenes as terminal end groups in marker dyes inbiologically relevant systems is so far not known in the art. This isdue to the extreme sensitivity to hydrolysis of these pi-deficientaromatics, especially in an aqueous basic environment (H. Lietz, G.Haucke, P. Czerney, B. John, J. Prakt. Chem., 1996, 338, 725-730).

Telfer et al. (U.S. Pat. No. 5,262,549) describe symmetrical trimethinesbased on 2-alkyl substituted benzopyrylium salts for the use as NIRabsorbers in polimeric media with a reduced tendency towards aggregationin these media.

OBJECT AND SUMMARY OF THE INVENTION

The primary object of the invention is to create NIR marker dyes basedon polymethine which have a high degree of photostability and stabilityin storage as well as a high fluorescent yield and which can be excitedto fluorescence in the easiest possible manner by means of laserradiation in the longwave visible or NIR spectral range, particularlywith light of a helium/neon or diode laser.

The present invention describes marker dyes based on non-symmetricalpolymethines which contain a substitutedω-(benz[b]pyran-4-ylidene)alk-1-enyl) unit of general formula (I),

where Z is a substituted derivative of benzooxazol, benzothiazol,2,3,3-trimethylindolenine, 2,3,3-trimethyl-4,5-benzo3H-indolenine, 3-and 4-picoline, lepidine, chinaldine and 9-methylacridine derivativeswith the general formulae IIa or IIb or IIc

and where

X stands for an element of the group O, S, Se or the structural elementN-alkyl or C(alkyl)₂,

N stands for the numerical value 1, 2 or 3,

R¹-R¹⁴ are equal or different and can be hydrogen, one or more alkyl,aryl, heteroaryl or heterocycloalipathic fragments, a hydroxy or alkoxygroup, an alkylsubstituted or cyclical amine function and/or twofragments in ortho position to each other, for example R¹⁰ and R¹¹, cantogether form another aromatic ring,

At least one of the substituents R¹-R¹⁴ can be a solubilizing orionizable or ionized substituent, like cyclodextrine, sugar, SO₃ ⁻, PO₃²⁻, COO⁻, or NR₃ ⁺, which determines the hydrophilic properties of thesedyes; here it is possible that this substituent can be bound to themarker dye by means of a spacer group,

At least one of the substituents R¹-R¹⁴ can stand for a reactive groupwhich facilitates a covalent linking of the dye to the aforementionedcarrier molecules, while this substituent can also be bound to the dyeby means of a spacer group, and

R¹ is a substituent which has a quarternary C-atom in alpha-positionrelative to the pyran ring. Examples for such substituents are t-butyl(—C(CH₃)₃) and adamantyl (—C₁₀H₁₅/tricyclo[3.3.1.1^(3,7)]decyl).

Subclaims 2 to 20 list specific embodiment forms and applications of themarker dyes.

These substituted derivatives of indol, heteroindol, pyridine, chinolineor acridine of the general formula I can be used as dyes for the opticalmarking of organic or inorganic microparticles, for example of proteins,nucleic acids, DNA, sugars, biological cells, lipids, drugs or organicor inorganic polymeric carrier substances.

Here, the marking of particles can be done by the formation of ionicinteraction between the markers of general formula I and the substancesto be marked.

The functional groups of these markers activated with regards tonucleophiles can couple covalently with an OH, NH₂ or SH function, whichtherefore creates a system for the qualitative and quantitativedetermination of organic and inorganic substances, like said proteins,nucleic acids, DNA, sugars, biological cells, lipids, drugs or organicor inorganic polymers.

The coupling reaction can take place in an aqueous or mostly aqueoussolution, preferably at room temperature. During this a conjugate withfluorescent properties is created.

By means of the preparation of non-symmetrical polymethines, which onthe one hand have an easily derivatizable heterocycle of the type of thepyridine, chinoline, indol, heteroindol or acridine derivatives and onthe other hand have a novel 6-ring heterocycle, in particular thefollowing advantages are achieved:

Trimethines already absorb in the spectral range >650 nm and have asignificantly improved photochemical and thermal stability when comparedwith polymethines known so far in the art which have absorptionmaxima >650 nm (penta- and heptamethines).

By means of molecular engineering, it is possible to control theposition and intensity of the absorption and emission maxima at will andto adapt them to emission wavelengths of different excitation lasers, inparticular NIR laser diodes.

The marker dyes can be produced by a relatively simple two-stagesynthesis with which a variety of dyes with functionalities that differ,for example, with regards to the total charge of the dye and the number,specificity and reactivity of the activated group used for theimmobilization can be provided in a manner that is specific to therespective application.

Compounds with the general formula I as well as systems derived fromthem (conjugates) can be used in optical, in particular fluorescenceoptical qualitative and quantitative determination methods for thediagnosis of cell properties, in biosensors (point-of-caremeasurements), exploration of the genome and in miniaturizationtechnology. Typical applications lie in the fields of cytometry, cellsorting, fluorescence correlation spectroscopy (FCS), ultra-highthroughput screening (UHTS), multicolor fluorescence in situhybridization (FISH) and in microarrays (gene and protein chips).

Here, a microarray is a grid-like arrangement of molecules immobilizedon at least one surface which can be used for the study of theinteraction between receptors and ligands. A grid-like arrangement meansmore than two molecules which are different from each other and whichare immobilized in different, predefined regions of known positions on asurface.

A receptor is a molecule which has an affinity to a given ligand.Receptors can be naturally occurring or artificially produced molecules.

Receptors can be used in their pure state or bound to other species.Receptors can be bound covalently or non-covalently either directly orvia certain coupling mediators to a bonding partner.

Examples of receptors which can be detected by means of this inventioninclude agonists and antagonists for cell membrane receptors, toxins andother poisonous substances, viral epitopes, hormones like opiates andsteroids, hormone receptors, peptides, enzymes, enzyme substrates,active substances that act as co-factors, lectines, sugars,oligonucleotides, nucleic acids, oligosaccharides, cells, cellfragments, tissue fragments, proteins and antibodies, but they are notlimited to the named substances.

A ligand is a molecule that is recognized by a particular receptor.Examples of ligands which can be detected by this invention includeagonists and antagonists for cell membrane receptors, toxins and otherpoisonous substances, viral epitopes, hormones like opiates andsteroids, hormone receptors, peptides, enzymes, enzyme substrates,active substances that act as co-factors, lectines, sugars,oligonucleotides, nucleic acids, oligosaccharides, cells, cellfragments, tissue fragments, proteins and antibodies, but they are notlimited to the named substances.

The invention is subsequently to be illustrated in more detail by meansof embodiment examples and the drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

In the accompanying drawings:

FIG. 1 shows the structural formula of benzopyrylium salt 2a;

FIG. 2 shows the synthesis and structural formula of benzopyrylium salt2b;

FIG. 3 shows the synthesis and structural formula of trimethine OB11(DY-630);

FIG. 4 shows the fluorescent spectra of OB 15 (DY-635) in an aqueoussolution and bound to bovine serum albumin (BSA); and

FIG. 5 shows the fluorescent excitation spectra of OB15 (DY-635) in anaqueous solution and bound to bovine serum albumin (BSA).

DESCRIPTION OF THE PREFERRED EMBODIMENTS Embodiment Examples

1. Instruction for the Preparation of11-(2,2-dimethylethyl)-9-methyl-1H,2H,3H,5H,6H,7H-pyrano[2,3-f]pyrido[3,2,1-ij]chinolin-12-iumtetrafluoroborate 2b (BS28), cf. FIG. 2:

50 ml of a 1.0 molar solution of methylmagnesiumbromide in dibutyletherare added drop by drop to a cooled solution of 7.3 g (0.0245 mol)11-(2,2-dimethylethyl)-1H,2H,3H,5H,6H,7H-pyrano[2,3-f]pyrido[3,2,1-ij]chinolin-9-onin 50 ml ethylenglycol-dimethylether. The mixture was heated to atemperature of 40 degrees C. for a time span of 30 minutes. Aftercooling down to 0 degrees C., 70 ml of a saturated NH₄Cl solution anddiluted hydrochloric acid were added for hydrolysis. The organic phasewas separated and extracted using 4×10 ml diethylether. The solvent wasremoved in a rotary evaporator and the oily residue was dissolved in 20ml pure acetic acid. The addition of 3 ml HBF₄ (48-50%) and the dilutionwith diethylether created a precipitant which is filtered out andrecrystallized from pure acetic acid.

A yield of 3.35 g (35%), melting point 175-180 degrees C.—¹H NMR (400MHz, CDCl₃+CF₃CO₂D): 1.43 (s, 9H), 1.90 (m, 2H), 2.06 (m, 2H), 2.67 (m,2H), 2.92 (m, 2H), 3.35 (m, 2H), 3.57 (m, 2H), 3.95 (s, 3H), 6.90 (s,1H), 7.58 (s, 1H):—C₂₀H₂₆BF₄NO (383.24): calculated C 62.68, H 6.84, N3.65, found C 63.06, H 6.72, N 3.48.

2. General Instruction for the Preparation of the Non-symmetricalTrimethines OB11, OB14, OB15 and OB20:

0.01 mol of the corresponding 4-methyl-benzopyrylium-tetrafluoroborateeaccording to formula 2a (BS4) or 2b (BS28) (cf. FIG. 1 and 2) and 0.01mol methylene-active N-heterocycle were dissolved in 20 ml acetanhydrideand after the addition of 2.0 g of triethoxymethane and 5 ml pyridineheated for about 10 minutes. The crude dye product was precipitated with30 ml of diethylether after the solution had cooled down to roomtemperature. The precipitate was filtered out and purified by means ofcolumn chromatography.

3.1-(5-carboxypentyl)-3,3-dimethyl-2-[3-(7-N,N-diethylamino-2-(1,1-dimethylethyl)-4H-benzopyran-4-ylidene)-1-propenyl]-3H-indolium-5-sulfonateOB11 (DY-630):

0.01 mol of 2a and 0.01 mol of1-(5-carboxypentyl)-2,3,3-trimethyl-3H-indolium-5-sulfonate weretransformed according to the general specification 1, see FIG. 3. Columnchromatography: SiO₂, eluent ethanol. Yield of 3.2 g (50%), meltingpoint 280-282 degrees C.—¹H NMR (400 MHz, DMSO-d6): 1.10-1.86 (m, 27H),2.16 (m, 2H), 3.54 (m, 4H), 4.13 (m, 2H), 6.58 (d, 1H), 6.74 (s, 1H),6.97 (s, 1H), 7.06 (d, 1H), 7.14 (d, 1H), 7.36 (d, 1H), 7.68 (d, 1H),7.78 (s, 1H), 8.08 (d, 1H), 8.32 (t, 1H)—¹³C NMR (100 MHz, DMSO-d6):12.30, 21.51, 24.32, 25.66, 26.56, 27.55, 34.07, 36.37, 43.72, 44.22,48.87, 96,36, 99.40, 104.11, 109.85, 110.28, 112.48, 113.27, 119.66,126.09, 140.23, 141.81, 145.59, 147.09, 162.14, 172.33, 174.64—MS (FABin dmba): 657 (M+Na⁺), 635 (M+H⁺), 391, 359, 258, 257—C₃₆H₄₆N₂O₆S(634.83): calculated C 68.11, H 7.30, N 4.41, found C 68.25, H7.33, N4.39.

4.1-(3-hydroxypropyl)-4-[3-(7-N,N-diethylamino-2-(1,1-dimethylethyl)-4H-benzopyran-4-ylidene)-1-propenyl]-chinolinium-tetrafluoroborateOB 14:

0.01 mol of 2a and 0.01 mol of1-(3-hydroxypopyl)-4-methylchinolinium-iodide were transformed accordingto general specification 1. Column chromatography: SiO₂, eluenttoluol/ethanol 1/1. Yield of 2.4 g (42%), melting point 162-164 degreesC.—¹H NMR (400 MHz, CDCl₃): 1.17 (t, 6H), 1.32 (s, 9H), 2.14 (m, 2H),2.25 (s, 1H), 3.39 (q, 4H), 3.71 (m, 2H), 4.89 (m, 2H), 6.31 (d, 1H),6.56 (s, 1H), 6.62 (m, 2H), 7.01 (d, 1H), 7.60 (t, 1H), 7.67 (d, 1H),7.77 (d, 1H), 7.84 (t, 1H), 7.93 (d, 1H), 8.12 (t, 1H), 8.31 (d, 1H),9.27 (d, 1H).—¹³C NMR (100 MHz, CDCl₃): 12.52, 28.08, 32.01, 36.20,44.61, 52.53, 57.52, 97.05, 97.94, 109.58, 109.94, 110.91, 111.77,113.61, 117.72, 124.79, 125.38, 125.50, 127.13, 133.64, 137.96, 140.92,142.20, 144.96, 150.87, 151.74, 155.40, 167.12—MS (FAB in dmba): 483(M⁺)—C₃₂H₃₉BF₄N₂O₂ (570.48): calculated C 67.37, H 6.89, N4.91, found C67.30, H 6.92, N 4.89.

5.1-(5-carboxypentyl)-3,3-dimethyl-2-[3-(11-(2,2-dimethylethyl)-1H,2H,3H,5H,6H,7H-pyrano[2,3-f]pyrido[3,2,1-ij]chinoline-9-ylidene)-1-propenyl]-3H-indolium-5-sulfonateOB 15 (DY-635):

0.01 mol of 2b and 0.01 mol of1-(5-carboxypentyl)-2,3,3-trimethyl-3H-indolium-5-sulfonate weretransformed according to the general specification 1.

Column chromatography: SiO₂, eluent ethanol. Yield of 2.9 g (44%),melting point >300 degrees C.—¹H NMR (250 MHz, DMSO-d6): 1.10-1.56 (m,19H), 1.91 (m, 4H), 2.08 (m, 4H), 2.83 (m, 4H), 3.38 (m, 4H), 4.03 (m,2H), 6.45 (d, 1H), 6.97 (s, 1H), 7.13 (d, 1H), 7.26 (d, 1H), 7.62 (d,1H), 7.73 (s, 1H), 7.78 (s, 1H), 8.23 (t, 1H)—¹³C NMR (62 MHz, DMSO-d6):19.40, 20.43, 24.86, 25.98, 26.61, 27.16, 27.76, 27.85, 28.94, 35.17,36.71, 43.40, 48.45, 49.04, 49.63, 99.24, 102.90, 105.09, 109.69,110.03, 112.96, 119.71, 121.85, 123.50, 139.89, 142.18, 144.84, 145.76,148.56, 148.86, 151.59, 170.08, 171.37—MS (ESI): 681 (M+Na⁺), 659(M+H⁺), 352—C₃₈H₄₆N₂O₆S (658.12): calculated C 69.27, H 7.34, N 4.25,found C 69.20, H 7.37, N 4.29.

6.1-(5-carboxypentyl)-4-[3-(7-N,N-diethylamino-2-(1,1-dimethylethyl)-4H-benzo-pyran-4-ylidene)-1-propenyl]-chinolinium-6-sulfonateOB20:

0.01 mol of 2a and 0.01 mol1-(5-carboxypentyl)-4-methylchinolinium-6-sulfonate were transformedaccording to general specification 1.

Column chromatography: SiO₂, eluent ethanol. Yield of 2.1 g (35%),melting point >300 degrees C.—C₃₅H₄₂N₂O₆S (618.76): calculated C 67.93,H 6.84, N 4.53, found C 67.73, H 6.93, N 4.29.

7. Preparation of the NHS Ester of OB11 (DY-630) withN-hydroxysuccinimide (NHS)/N,N′-dicylco-hexylcarbodiimide (DCC)

15 mg OB11 (DY-630), 14 mg DCC and 4 mg NHS were dissolved in 1 ml dryDMF. After this, 1 μl of thriethylamine were added. The reaction mixturewas stirred for 24 hours at room temperature and then filtered. Thesolvent was then drawn off, the residue was washed with ether. Thisreaction was quantitative.

8. Preparation of the NHS Ester of OB15 (DY-635) withN-hydroxysuccinimide (NHS)/N,N′-dicylco-hexylcarbodiimide (DCC)

The process was analogous to example 7. This reaction also wasquantitative.

9. Excitation and Emission Spectra of1-(5-carboxypentyl)-3,3-dimethyl-2-[3-(11-(2,2-dimethylethyl)-1H,2H,3H,5H,6H,7H-pyrano[2,3-f]pyrido[3,2,1-ij]chinoline-9-ylidene)-1-propenyl]-3H-indolium-5-sulfonateOB15 (DY-635)

The diagram in FIG. 4 shows the emissions spectra and the diagram FIG. 5shows the excitation spectra of1-(5-carboxypentyl)-3,3-dimethyl-2-[3-(11-(2,2-dimethylethyl)-1H,2H,3H,5H,6H,7H-pyrano[2,3-f]pyrido[3,2,1-ij]chinolin-9-ylidene)-1-propenyl]-3H-indolium-5-sulfonatewhen in water and when non-covalently bound to bovine serum albumin(BSA), with the more intense spectrum being the one of the BSAconjugate. The concentration of both dye solutions was identical forthese measurements.

10. General Specification for the Marking of Proteins

The protein marking was done in a 50 mM bicarbonate buffer (pH 9.0). Aparent solution with 0.5 mg reactive dye (for example OB11-NHS-ester,M=732 g*mol⁻¹) in 100 μl DMF was created. The protein, for exampleavidine (M=66000 g*mol⁻¹) was dissolved step by step in portions of 1 mgin 200 ml bicarbonate buffer; after this, varying volumes of thedifferent and—if necessary—diluted dye parent solutions were added tothe different protein aliquots. The reaction mixtures were then stirredfor one to two hours at room temperature. The free dye was separatedfrom the marked proteins by means of gel chromatography (Sephadex G25medium, eluent PBS pH 7.2 22 mM).

While the foregoing description and drawings represent the presentinvention, it will be obvious to those skilled in the art that variouschanges may be made therein without departing from the true spirit andscope of the present invention.

What is claimed is:
 1. Laser-compatible NIR marker dyes based onpolymethines of the general formula I, II or III

wherein R¹ to R¹⁴ are equal or different and are present in each casehydrogen, chlorine, bromine, an aliphatic or mononuclear aromatic group,each having at most 12 carbon atoms which may contain as a substitutedgroup in addition to carbon and hydrogen, up to 4 oxygen atoms and zero,one or two nitrogen atoms or a sulfur atom or a sulfur and a nitrogenatom or represent an amino function, having a nitrogen atom to whichthere is bound, hydrogen or at least one substituent having up to 8carbon atoms, said substituent selected from the group consisting ofcarbon, hydrogen and up to two sulfonic acid groups.
 2. The marker dyeof claim 1, wherein at least one of the groups R¹ to R¹⁴ contains asolubilizing or ionizable group.
 3. The marker dye of claim 2, whereinsaid solubilizing or ionizable group is bound via an aliphatic orheteroallphatic group.
 4. The marker dye of claim 2, wherein thesolubilizing or ionizable group is SO⁻³, CO₂H, OH or a combinationthereof.
 5. The marker dye of claim 1, wherein at least one of the saidgroups R¹ to R¹⁴ contains a reactive group which is capable of reactingwith a biomolecule to form a covalent bond.
 6. The marker dye claim 5,wherein the reactive group is a N-hydroxysuccinimide ester group or amaleimide group or a phosphoramidite group.
 7. The marker dye of claim5, wherein any of the groups R¹ to R¹⁴ which is allphatic and containsform 1 to 6 carbon atoms.
 8. The marker dye of claim 1, wherein nrepresents zero, 1 or 2.